1. C - 1 PowerPoint to accompany Genetics: From Genes to Genomes Fourth Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver Reference C Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Prepared by Malcolm Schug University of North Carolina Greensboro

2. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 2 Caenorhabditis elegans: Genetic Portrait of a Simple Multicellular Animal

3. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 3 Fig. C.1

4. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 4 Summary of Reference C Overview of C. elegans as experimental organism Overview of C. elegans as experimental organism Description of genome Description of genome Life cycle and anatomy Life cycle and anatomy Precise patterns of cell lineage throughout development Precise patterns of cell lineage throughout development Techniques of genetic and molecular analysis Techniques of genetic and molecular analysis Genetic dissection of several developmental processes Genetic dissection of several developmental processes Specification of early embryonic blastoderms Specification of early embryonic blastoderms Role of programmed cell death Role of programmed cell death Timing of decision during larval development Timing of decision during larval development Comprehensive example on use of genetics to probe a signaling pathway that helps control development of the hermaphrodite vulva Comprehensive example on use of genetics to probe a signaling pathway that helps control development of the hermaphrodite vulva

5. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 5 Nuclear Genome of C. elegans 97 Mb – very small (2/3 size of Drosophila) 97 Mb – very small (2/3 size of Drosophila) Six chromosomes (I-VI) – about same size Six chromosomes (I-VI) – about same size No defined centromere – holocentric No defined centromere – holocentric Detailed physical map of cosmids and YACs Detailed physical map of cosmids and YACs Recombination rates vary considerably Recombination rates vary considerably 1 gene every 5 kb 1 gene every 5 kb 19,000 genes 19,000 genes Proteins match homologous sequences of other organisms Proteins match homologous sequences of other organisms 20% proteins carry out core biological functions 20% proteins carry out core biological functions Rest involved in processes required only in multicellular organisms Rest involved in processes required only in multicellular organisms Repetitive sequences – e.g., 7 kinds of TEs Repetitive sequences – e.g., 7 kinds of TEs

6. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 6 Gene Expression 70% of mRNAs have one of two splice- leader sequences trans-spliced onto the 5 end of the message 70% of mRNAs have one of two splice- leader sequences trans-spliced onto the 5 end of the message Conserved among all nematode genera examined Conserved among all nematode genera examined 25% of adjacent genes are transcribed as operons 25% of adjacent genes are transcribed as operons Trans-splicing with splice-leader sequences produces single-gene mRNAs Trans-splicing with splice-leader sequences produces single-gene mRNAs

7. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 7 Fig. C.3 Gene expression

8. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 8 Life cycle of C. elegans Hermaphrodite Hermaphrodite First 40 germ cells that enter meiosis in each arm of gonad develop into ~150 sperm deposited into spermatheca First 40 germ cells that enter meiosis in each arm of gonad develop into ~150 sperm deposited into spermatheca Sexual fate switches and rest of germ cells are oocytes Sexual fate switches and rest of germ cells are oocytes Fig. C. 4

9. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 9 Mating Male lies next to female and slides fan-shaped tail along its body until it contacts the vulva Male lies next to female and slides fan-shaped tail along its body until it contacts the vulva Inserts specialized structures at base of its tail into vulva Inserts specialized structures at base of its tail into vulva Sperm move from male’s tail through vulva and into uterus Sperm move from male’s tail through vulva and into uterus Sperm then migrate to spermathecae Sperm then migrate to spermathecae Gain advantage over resident sperm and produce outcrossed progeny Gain advantage over resident sperm and produce outcrossed progeny

10. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 10 Development from Zygote to Adult 3 days 3 days Embryonic development begins in uterus Embryonic development begins in uterus Hermaphrodite lays her eggs 2 hours after fertilization Hermaphrodite lays her eggs 2 hours after fertilization Blastoderms – large cells produced by early cleavage Blastoderms – large cells produced by early cleavage Unequal cleavages in early development Unequal cleavages in early development Until 28 cell stage all blastoderms contact surface of embryo Until 28 cell stage all blastoderms contact surface of embryo Gastrulation - At 28 cell stage, two gut precursor cells begin to move to embryo interior Gastrulation - At 28 cell stage, two gut precursor cells begin to move to embryo interior Three germ layers produced (endoderm, mesoderm, ectoderm) Three germ layers produced (endoderm, mesoderm, ectoderm) Later, cell division ceases while cells differentiate Later, cell division ceases while cells differentiate Contractile proteins squeeze embryo into wormlike shape Contractile proteins squeeze embryo into wormlike shape 14 hours after fertilization, egg hatches 14 hours after fertilization, egg hatches L1 larvae is 250mm long and has 558 cells L1 larvae is 250mm long and has 558 cells Next 50 hours – L2, L3, L4 stages of development separated by molts Next 50 hours – L2, L3, L4 stages of development separated by molts

11. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 11 Figure C.5a Embryonic Development in C. elegans Unequal cleavage gastrulationwormlike shape before hatching

12. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 12 Fig. C.5b C. elegans Life Cycle

13. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 13 Laboratory Culture Grow on agar plates with lawn of E. coli for food Grow on agar plates with lawn of E. coli for food Transfer with flattened platinum wire Transfer with flattened platinum wire Adults reproduce for 3-4 days Adults reproduce for 3-4 days Adults live up to 2 weeks after reproduction Adults live up to 2 weeks after reproduction Dauer larva Dauer larva Arrest development at L3 if starved Arrest development at L3 if starved Can live up to 6 months Can live up to 6 months Given food they reinitiate development Given food they reinitiate development Can arrest aging in response to food deprivation Can arrest aging in response to food deprivation

14. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 14 Anatomy of an Adult Cross section Fig. C.6a

15. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 15 Linear Diagrams Show the Life History of Every Somatic Cell Researchers focus on small part of cell lineage to study particular developmental process Researchers focus on small part of cell lineage to study particular developmental process Partially metameric Partially metameric Composed of repeating patterns of cell groups with similar functions Composed of repeating patterns of cell groups with similar functions Fig. C.6b

16. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 16 Cell Lineage Diagram Fig. C.7a

17. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 17 Lineage Diagram of Early Cleavages Give Rise to Six Founder Cells Which Each Give Rise to Various Features of Adult

18. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 18 Genetic Analysis and Recombinant DNA Technology Diploid organism Diploid organism Males have single X chromosome (XO) Males have single X chromosome (XO) Hermaphrodites are XX Hermaphrodites are XX Males arise spontaneously via nondisjunction of X during hermaphrodite gametogenesis Males arise spontaneously via nondisjunction of X during hermaphrodite gametogenesis Once males present, mating generates more Once males present, mating generates more After fertilization, half gametes result from sperm that carry no X = XO males After fertilization, half gametes result from sperm that carry no X = XO males

19. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 19 Mutant Isolation EMS induces high forward mutation rate EMS induces high forward mutation rate Hermaphrodite heterozygotes for mutation will produce Hermaphrodite heterozygotes for mutation will produce ¼ homozygous mutant progeny by self-fertilization Severe effect mutations recovered because of self- fertilization (e.g., complete paralysis of body wall muscles) Severe effect mutations recovered because of self- fertilization (e.g., complete paralysis of body wall muscles) 1600 genes on genetic map from mutant screens 1600 genes on genetic map from mutant screens Examples Examples Dumpy (Dy) – abnormal body shape Dumpy (Dy) – abnormal body shape Long (Lon) – abnormal body shape Long (Lon) – abnormal body shape Unc – uncoordinated Unc – uncoordinated Phenotypes capitalized and not italicized Phenotypes capitalized and not italicized Genes and alleles are in lower-case italics Genes and alleles are in lower-case italics Protein products are all in upper case (e.g., DPY-10) Protein products are all in upper case (e.g., DPY-10)

20. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 20 DNA Transformation Inject DNA into distal syncytial gonad of hermaphrodites Inject DNA into distal syncytial gonad of hermaphrodites Irradiation promotes integration of transgenes into genome Irradiation promotes integration of transgenes into genome Reporter constructs show transgenes Reporter constructs show transgenes GFP is commonly used reporter gene GFP is commonly used reporter gene Fig. C.8

21. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 21 RNAi Enzyme complex, dicer, cleaves dsRNA into 22bp fragments called trigger RNAs or small interfering RNAs (siRNAs) Enzyme complex, dicer, cleaves dsRNA into 22bp fragments called trigger RNAs or small interfering RNAs (siRNAs) siRNAs spread throughout cells siRNAs spread throughout cells siRNAs unwind and complex with complementary sequence of endogenous mRNA siRNAs unwind and complex with complementary sequence of endogenous mRNA two fates of siRNA two fates of siRNA Destruction of mRNA Destruction of mRNA Reverse transcribed, cleaved, more siRNA – amplification of siRNA Reverse transcribed, cleaved, more siRNA – amplification of siRNA Fig. C.9

22. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 22 Cloning of C. elegans Genes Positional cloning – based on location in genome Positional cloning – based on location in genome SNPs used as markers SNPs used as markers Identification of gene Identification of gene Rescue mutant phenotype by injection into hermaphrodites, each cosmid corresponding to interval identified by positional cloning Rescue mutant phenotype by injection into hermaphrodites, each cosmid corresponding to interval identified by positional cloning Candidate gene approach using complete genome sequence Candidate gene approach using complete genome sequence Candidate genes can be tested by RNAi Candidate genes can be tested by RNAi

23. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 23 Identification of Mutations Caused by Insertion of a Transposable Element Forward genetics Forward genetics Begin with mutant phenotype and isolate gene for molecular analysis Begin with mutant phenotype and isolate gene for molecular analysis Reverse genetics Reverse genetics Gene of possible developmental interest predicted from genomic DNA sequence Gene of possible developmental interest predicted from genomic DNA sequence Assign function through PCR methods Assign function through PCR methods Assign function through RNAi Assign function through RNAi Fig. C.10

24. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 24 Genetic Mosaic Analysis Animals composed of wild-type and mutant cells Animals composed of wild-type and mutant cells Helps determine focus of action for a gene Helps determine focus of action for a gene Cells that must be active to allow animal to develop normally Cells that must be active to allow animal to develop normally Chromosomal fragments generated from X or  irradiation Chromosomal fragments generated from X or  irradiation Fragments maintained as extrachromosomal free duplications in stocks Fragments maintained as extrachromosomal free duplications in stocks Construct strain chromosomally homozygous for recessive mutation Construct strain chromosomally homozygous for recessive mutation Introduce free duplication including wild-type allele of same gene Introduce free duplication including wild-type allele of same gene If free duplication is lost, clone of mutant cells will arise If free duplication is lost, clone of mutant cells will arise

25. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 25 A genetic mosaic experiment A genetic mosaic experiment Free duplication carries wild-type allele of lin-12 + in a mutant worm (lin-12 - ) Free duplication carries wild-type allele of lin-12 + in a mutant worm (lin-12 - ) mrk + on duplication, mrk - in background provides phenotypic marker indicating loss of lin-12 free duplication mrk + on duplication, mrk - in background provides phenotypic marker indicating loss of lin-12 free duplication Results of mosaic analysis showing Z1.ppp or Z4.aaa must have lin-12 + activity to assume a VU shape Results of mosaic analysis showing Z1.ppp or Z4.aaa must have lin-12 + activity to assume a VU shape Fig. C.11

26. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 26 C. Elegans Research in Postgenomic Era Functional genomics Functional genomics Identification of all the genes expressed and all genes required during various stages of development or in response to physiological stimulus Identification of all the genes expressed and all genes required during various stages of development or in response to physiological stimulus Global analysis using DNA chips with ordered microarrays of 19,000 predicted genes in genome Global analysis using DNA chips with ordered microarrays of 19,000 predicted genes in genome In situ hybridization patterns at various stages of development using probes for every predicted gene In situ hybridization patterns at various stages of development using probes for every predicted gene

27. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 27 Identification of Maternal Effect Lethals Specification of early embryonic blastoderms Specification of early embryonic blastoderms Early cleavages depend on maternally supplied components Early cleavages depend on maternally supplied components F1 hermaphrodites F1 hermaphrodites No vulva No vulva Progeny eat them from the inside Progeny eat them from the inside Surviving F1 have mutant progeny that arrest development before hatching Surviving F1 have mutant progeny that arrest development before hatching Fig. C.12

28. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 28 Cell-autonomous Determinants must be Distributed to Cells in Which They Act PAR proteins help direct embryonic polarity PAR proteins help direct embryonic polarity (a) wild-type zygotes, P granules initially uniformly distributed, but then localize to posterior part of zygote (a) wild-type zygotes, P granules initially uniformly distributed, but then localize to posterior part of zygote (b) shortly after fertilization, PAR-2 is found in posterior cortex and PAR-3 in anterior cortex (b) shortly after fertilization, PAR-2 is found in posterior cortex and PAR-3 in anterior cortex Maternal lethal mutations that produce two-cell embryos with no polarity identify six par genes Maternal lethal mutations that produce two-cell embryos with no polarity identify six par genes Fig. C.13

29. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 29 Inductive Ssignals Control Cell Fates in Early Embryogenesis PAR proteins only affect cells that contain them PAR proteins only affect cells that contain them Cell autonomous Cell autonomous Early development also depends on control signals sent between cells to partition Early development also depends on control signals sent between cells to partition Intercellular signaling determines fates of ABa and ABp cells Intercellular signaling determines fates of ABa and ABp cells Contact between P 2 and ABp enables P 2 to send signals to ABp required for proper fate of Abp lineage Contact between P 2 and ABp enables P 2 to send signals to ABp required for proper fate of Abp lineage Figure C.14

30. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 30 Programmed Cell Death - Apoptosis Fig. C.15

31. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 31 Control of Timing During Larval Development Heterochronic mutations Heterochronic mutations Result in inappropriate timing of cell division and cell-fate decisions during larval development Result in inappropriate timing of cell division and cell-fate decisions during larval development Switch genes – loss-of- function and gain-of- function mutations have opposite temporal effects Switch genes – loss-of- function and gain-of- function mutations have opposite temporal effects Lin-14 loss-of-function mutation cases retarded development Lin-14 loss-of-function mutation cases retarded development Alter timing and cell cycle Alter timing and cell cycle Fig. C.16

32. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 32 LIN-14 protein behaves as central clock LIN-14 protein behaves as central clock Concentration descends from high in L1 animals to a low in L4 animals Concentration descends from high in L1 animals to a low in L4 animals Epistasis tests show lin-4 negatively regulates expression of lin-14 by novel mechanism Epistasis tests show lin-4 negatively regulates expression of lin-14 by novel mechanism Lin-4 gene transcript is processed to produce a smaller 22-nucleotide RNA complementary to 3 untranslated transcript of lin-14 mRNA Lin-4 gene transcript is processed to produce a smaller 22-nucleotide RNA complementary to 3 untranslated transcript of lin-14 mRNA RNAi machinery appears to control LIN-14 concentrations RNAi machinery appears to control LIN-14 concentrations

33. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 33 Using Genetics to Probe Development of Hermaphrodite Vulva Mutant screens identify genes involved in vulva formation Mutant screens identify genes involved in vulva formation 6 cells capable of giving rise to vulva (P3p – P8p) 6 cells capable of giving rise to vulva (P3p – P8p) P5p-P7p normally give rise to vulva in response to inductive signal from anchor cell (AC) P5p-P7p normally give rise to vulva in response to inductive signal from anchor cell (AC) P3p, P4p, P8p produce only additional hypodermal cells P3p, P4p, P8p produce only additional hypodermal cells Fig. C.17a

34. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 34 Genetic screens identify genes involved in AC induction of P5p, P6p, and P7p Genetic screens identify genes involved in AC induction of P5p, P6p, and P7p Search for vulvaless mutants Search for vulvaless mutants Mutants with no vulva fertilize eggs, eggs hatch and devour mother’s organs Mutants with no vulva fertilize eggs, eggs hatch and devour mother’s organs Multivulva mutants form ectopic vulva-like structures Multivulva mutants form ectopic vulva-like structures Fig. C.17b

35. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display C - 35 Signal Transduction Pathway Activated by Binding of LIN-3 Ligand to LET-23 Receptor Fig. C.17c